The regulation of aromatase and androgen receptor expression during gonad development in male and female European eel

[EN] This research investigated the regulation of aromatase and androgen receptor gene expression in the brain–pituitary– gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa-cyp19a1) and partial androgen receptor a and b (aa-ara and aa-arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa-arb was expressed at higher levels than aa-ara in the pituitary and gonad in both sexes, suggesting aa-arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa-ara and an increase in aa-cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa-arb and aa-cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa-arb and an increase in aa-cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa-cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex-dependent differences in the regulation of the expression of aa-ara, aa-arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturationThis work was funded by the European Community's 7th Framework Programme under the Theme 2 'Food, Agriculture and Fisheries, and Biotechnology', grant agreement no 245257 (PRO-EEL). D. S. P. received a postdoc grant from UPV (CEI-01-10), a mobility grant from UPV (PAID-00-11) and has also been supported by a contract cofinanced by MICINN and UPV (PTA2011-4948-I). V. G. and I. M. received predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN) and Generalitat Valenciana, respectively. F.-A. W. received funding from the Norwegian School of Veterinary Science. The fish farm Valenciana de Acuicultura, S. A. supplied the male eels used in the experiments. The English revision was carried out by Professor Lucy Robertson (Lucy Robertson Writing Services, Norway).Peñaranda, D.; Mazzeo, I.; Gallego Albiach, V.; Hildahl, J.; Nourizadeh-Lillabadi, R.; Pérez Igualada, LM.; Weltzien, FA.... (2014). The regulation of aromatase and androgen receptor expression during gonad development in male and female European eel. Reproduction in Domestic Animals. 49(3):512-521. https://doi.org/10.1111/rda.12321512521493Aroua, S., Weltzien, F.-A., Belle, N. L., & Dufour, S. 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Isolation of Atlantic halibut pituitary hormones by continuous-elution electrophoresis followed by fingerprint identification, and assessment of growth hormone content during larval development

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS–PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.We thank Heiðdís Smáradóttir and Arnar Jónsson at Fiskey Ltd., Iceland, for excellent logistic and scientific collaboration. This work has been carried out within the project ‘‘Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis’’ (Q5RS-2002-01192) with financial support from the Commission of the European Communities. However, it does not necessarily reflect the Commission’s views and in no way anticipates its future policy in this area. This project was further supported by the Swedish Council for Agricultural and Forestry Research (FORMAS) and Pluriannual funding to CCMAR by the Portuguese Science and Technology Council

Identification of two isoforms of Atlantic halibut insulin-like growth factor-I receptor genes and quantitative gene expression during metamorphosis

Insulin-like growth factor-I (IGF-I) is an important regulator of growth and development in vertebrates. Both the endocrine and paracrine actions of IGF-I are mediated through ligand-binding to a membrane-bound IGF-I receptor (IGF-IR). The characterization of this receptor and subsequent expression studies thus help elucidate the endocrine regulation of developmental processes. As other flatfish species, the Atlantic halibut (Hippoglossus hippoglossus) undergoes a dramatic larval metamorphosis. This process is largely under endocrine control, and data indicate that IGF-I could be a key regulator. IGF-I content increases up to late pre-metamorphosis and decreases during metamorphosis. The IGF-IR has, however, not been studied during flatfish metamorphosis. To examine IGF-IR gene expression, two IGF-IR mRNA were cloned and sequenced. These partial sequences share high identity (≥ 95%) and similarity (≥ 97%) with other fish IGF-IR and lower identity (≥ 77%) and similarity (≥ 83.5%) with Japanese flounder insulin receptors. The expression of mRNA for both IGF-IR was analyzed by quantitative real-time RT-PCR during six larval developmental stages from pre- to post-metamorphosis. IGF-IR1 and IGF-IR2 mRNA are differentially expressed during metamorphosis, but if this indicates an isoform-specific regulation of developmental processes by circulating and/or locally-secreted IGF-I is unclear. Both IGF-IR genes are down-regulated in halibut larvae experiencing arrested metamorphosis, suggesting the IGF-I system is critical for metamorphic success in halibut

Cloning of Atlantic halibut growth hormone receptor genes and quantitative gene expression during metamorphosis

To gain insight into the possible regulatory role of the growth hormone (GH)-insulin-like growth factor I (IGF-I) system in flatfish metamorphosis, body GHR gene expression as well as IGF-I protein content was quantified in larval Atlantic halibut throughout metamorphosis (developmental stages 5–10). The cDNA of the full-length GH receptor (hhGHR) was cloned from adult liver and characterized. The hhGHR shows common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and an intracellular domain containing a Box 1 and Box 2. Additionally, a truncated GHR (hhGHRtr), similar to turbot and Japanese flounder GHRtr, was cloned and sequenced. These sequences are highly similar to the full-length and truncated GHRs in turbot (89%/86%) and Japanese flounder (93%/91%) with lower identity with other fish type I GHR (⩽81%) and type II GHRs (⩽58%). A quantitative real-time RT-PCR assay was used to measure hhGHR and hhGHRtr mRNA content in normally and abnormally metamorphosed individuals at six developmental stages, from early pre-metamorphosis to post-metamorphosis, when the fish is considered a juvenile. The level of hhGHR gene expression was highest at pre-metamorphic stage 6 and at stage 8 at the onset of metamorphosis, and then decreased during metamorphic climax and post-metamorphosis. Expression of hhGHRtr reached highest levels at stage 6 and then decreased to post-metamorphosis. The ratio of expression between the full-length and the truncated GHR (hhGHR:hhGHRtr) varied among stages and was highest at the onset of metamorphosis and at metamorphic climax. A radioimmunoassay was used to measure halibut IGF-I body content throughout metamorphosis. IGF-I increases from early metamorphosis to the onset of metamorphosis and then decreases towards post-metamorphosis. In comparison between normally and abnormally metamorphosing larvae, IGF-I content, hhGHR and hhGHRtr mRNA levels were reduced in the abnormal fish. These data indicate that the GH-IGF-I system either has a regulatory role in metamorphosis, or is being affected as a consequence of the abnormal metamorphosis

Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation

[EN] Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out. An experimental group of silver-stage eel females was maintained at 18 C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12 weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development. These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation.Funded from the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", Grant agreement no. 245257 (Pro-Eel) and Generalitat Valenciana through the GV/2007/202 project and ACOMP/2011/229. D.S. Penaranda has a postdoc Grant from the Universitat Politecnica de Valencia (CE-01-10). I.M. and V.G. have predoctoral Grants from Generalitat Valenciana and Spanish MICINN, respectively. F.-A. Weltzien received funding from The Norwegian School of Veterinary Science.Mazzeo, I.; Peñaranda, D.; Gallego Albiach, V.; Hildahl, J.; Nourizadeh-Lillabadi, R.; Asturiano Nemesio, JF.; Pérez Igualada, LM.... (2012). Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation. General and Comparative Endocrinology. 178:338-346. https://doi.org/10.1016/j.ygcen.2012.06.003S33834617

Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development

[EN] Gonadotropin-releasing hormone receptor (GnRH-R) activation stimulates synthesis and release of gonadotropins in the vertebrate pituitary and also mediates other processes both in the brain and in peripheral tissues. To better understand the differential function of multiple GnRH-R paralogs, three GnRH-R genes (gnrhr1a, 1b, and 2) were isolated and characterized in the European eel. All three gnrhr genes were expressed in the brain and pituitary of pre-pubertal eels, and also in several peripheral tissues, notably gills and kidneys. During hormonally induced sexual maturation, pituitary expression of gnrhr1a (female) and gnrhr2 (male and female) was up-regulated in parallel with gonad development. In the brain, a clear regulation during maturation was seen only for gnrhr2 in the midbrain, with highest levels recorded during early vitellogenesis. These data suggest that GnRH-R2 is the likely hypophysiotropic GnRH-R in male eel, while both GnRH-R1a and GnRH-R2 seems to play this role in female eels.aa-gnrhr2 Primers were obtained from a partial sequence kindly provided by Dr. Kataaki Okubo (University of Tokyo, Japan). The fish farm Valenciana de Acuicultura, S.A. supplied the male fish used in the described experiments. This work was funded by the European Community's 7th Framework Program under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", Grant Agreement No. 245257 (Pro-Eel) and Generalitat Valenciana (ACOMP/2012/086 and I.M. Predoctoral Grant). D.S.P. has a postdoc Grant from UPV (CEI-01-10), mobility grants from UPV (PAID-00-11) and the Research Council of Norway (EJ/hsm IS-STP, 2009) and also has been supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I). V.G. and I.M. have predoctoral grants from Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), while F.-A.W. was funded by the Norwegian School of Veterinary Science.Peñaranda, D.; Mazzeo, I.; Hildahl, J.; Gallego Albiach, V.; Nourizadeh-Lillabadi, R.; Pérez Igualada, LM.; Asturiano Nemesio, JF.... (2013). Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development. Molecular and Cellular Endocrinology. 369(1):1-14. https://doi.org/10.1016/j.mce.2013.01.025S114369

The molecular and endocrine basis of flatfish metamorphosis

A significant component of aquaculture is the production of good quality larvae, and, in the case of flatfish, this is tied up with the change from a symmetric larva to an asymmetric juvenile. Despite the pioneering work carried out on the metamorphosis of the Japanese flounder (Paralichthys olivaceus) and summer flounder (Paralichthys dentatus), the underlying molecular basis of flatfish metamorphosis is still relatively poorly characterized. It is a thyroid hormone (TH) driven process, and the role of other hormones in the regulation of the process along with the interplay of abiotic factors are still relatively poorly characterized as is the extent of tissue and organ remodeling, which underlie the profound structural and functional modifications that accompany the larval/juvenile transition. The isolation of genes for hormones, receptors, binding proteins, and other accessory factors has provided powerful tools with which to pursue this question. The application of molecular methodologies such as candidate gene approaches and microarray analysis coupled to functional genomics has started to contribute to understanding the complexity of tissue and organ modifications that accompany flatfish metamorphosis. A better understanding of the biology of normal metamorphosis is essential to identify factors contributing to abnormal metamorphosis.The present invited review was prepared in the context of the European Specific Support Action, AQUAFUNC (EU-SSA- 022685) with financial support from the Commission of the European Communities. We thank the anonymous reviewers whose comments and suggestions have contributed to improve the manuscript. Some of the work reported was carried out in a number of projects: “Arrested Development: The Molecular and Endocrine Basis of Flatfish Metamorphosis” (ARRDE; Q5RS- 2002-01192) with financial support from the Commission of the European Communities; the Swedish Council for Agricultural and Forestry Research (FORMAS) and Plurianual funding to CCMAR from the Portuguese Science and Technology Council